CROSS-REFERENCE TO RELATED APPLICATIONS
STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with United States government support awarded by the following
agency: NIH grant 1-U54-AI-057153. The United States government has certain rights
in this invention.
BACKGROUND OF THE DISCLOSURE
[0003] Clostridium botulinum produces seven different neurotoxins (BoNTs) which differentiated
serologically by the lack of anti-serum cross serotype neutralization. BoNTs are the
most potent toxins known to humans and are the causative agents of the disease botulism
(1). BoNTs exert their action by inhibiting the release of the neurotransmitter acetylcholine
at the neuromuscular junction, leading to a state of flaccid paralysis. BoNTs elicit
neuronal-specific flaccid paralysis by targeting neurons and cleaving neuron specific
SNARE proteins.
[0004] SNARE proteins (Soluble NSF Attachment protein Receptors) are a large superfamily
of proteins. The main function of SNARE proteins is to mediate the exocytosis of neurotransmitter
molecules to the post-synaptic junction. SNAREs are small, abundant and both vesicle
and plasma-membrane bound proteins.
[0005] BoNTs are a 150kDa polypeptide chain comprising a 100kDa heavy chain and a 50kDa
light chain linked by a disulfide bond. BoNTs are organized into three functional
domains: an N-terminal zinc metalloprotease light chain (LC), a translocation domain
(HCT) and a C-terminal receptor binding domain (HCR) (1, 2). The toxic effect of BoNTs
(nerve intoxification) is accomplished through the interplay of three key events.
One, the carboxy half of the heavy chain is required for receptor-specific binding
to cholinergic nerve cells at the nerve-cell membrane. After binding, another portion
of the BoNT moves a smaller catalytic domain into the cell, where the catalytic domain
binds to and cleaves a neuronal SNARE protein, "intoxicating" the nerve cell, making
it impossible to "fire" or send signals. By "catalytic domain" we mean the part of
the molecule that triggers the cleavage of the substrate. The toxin is internalized
into an endosome through receptor-mediated endocytosis, and the toxin binds the liminal
domains of synaptic vesicle-associated proteins upon the fusion of synaptic vesicles
with the plasma membrane (3-5). In short, BoNTs are internalized into endosomes and
upon acidification, the LC is translocated into the cytoplasm, where SNARE proteins
are cleaved (1, 2).
[0006] Mammalian neuronal exocytosis is driven by the formation of protein complexes between
the vesicle SNARE, VAMP2, and the plasma member and SNAREs, SNAP 25 and syntaxin 1a
(6). There are seven serotypes of BoNTs (termed A-G) that cleave specific residues
on one of three SNARE proteins: serotypes B, D, F, and G cleave VAMP-2, serotypes
A and E cleave SNAP 25, and serotype C cleaves SNAP 25 and syntaxis 1a (1). Thus,
neuronal specificity is based upon BoNT binding to neurons and cleaving neuronal isoforms
of the SNARE proteins. For example, BoNT/A cleaves human SNAP25, but not the human
non-neuronal isoform SNAP 23 (7, 8). The non-neuronal SNARE isoforms are involved
in a divergent cellular processes, including fusion reactions in cell growth, membrane
repair, cytokinesis and synaptic transmission.
[0007] The reversible nature of muscle function after BoNTs intoxication that replace toxin-affected
nerves with new nerves (10, 11), has turned the BoNTs from a deadly agent to novel
therapies for neuromuscular conditions. As early as 1989, BoNT/A was approved by the
FDA to treat strabismus, blepharospasm, and hemifacial spasm and then for cervical
dystonia, cosmetic use, glabellar facial lines and axillary hyperhidrosis (12). BoNT/A
efficacy in dystonia and other disorders related to involuntary skeletal muscle activity,
coupled with a satisfactory safety profile, and prompted empirical/off-label use in
a variety of secretions and pain and cosmetic disorders (13).
[0008] The clinical use of BoNTs is limited to targeting inflictions affecting neuromuscular
activity (12, 13). Elucidation of the structure-function relationship of BoNTs has
enabled the design of novel therapies that retarget BoNT to unique neurons and non-neuronal
cells. Replacement of BoNT HCR domain with nerve growth factor, lectin from Erythrina
cristagalli, or epidermal growth factors enable retargeting of BoNT/A to neuronal
or non-neuronal cells such as nociceptive afferents and airway epithelium cells (14-16).
However, the selective cleavage of neuronal specific SNARE proteins by BoNT has limited
development of novel therapies in these non-neuronal systems. Prerequisite to develop
novel therapies requires the retargeting of the catalytic activity of the BoNTs to
non-neuronal SNARE isoforms.
[0009] Accordingly, a need exists for an engineered BoNT that cleaves non-neuronal SNARE
proteins and methods of use thereof.
SUMMARY
[0010] The invention is defined by the claims. Those aspects/instances of the present disclosure
which constitute the invention are defined by the claims.
[0011] In one instance, the present disclosure provides a modified BoNT/E catalytic domain,
wherein light chain residue 224, or a residue corresponding to residue 224, has been
altered. In one instance, the residue 224 has been altered to be aspartic acid or
glutamic acid. The modified catalytic domain cleaves SNAP23 but does not cleave SNAP29
or SNAP47.
[0012] In an alternate instance, the modified catalytic domain additionally comprise a target
molecule useful in a protein delivery system.
[0013] In an alternate instance, the present disclosure provides an engineered botulinum
neurotoxin E or botulinum neurotoxin light chain or botulinum toxin catalytic domain
comprising a modified BoNT/E catalytic domain, wherein light chain residue 224 has
been altered.
[0014] In an alternate instance, the present disclosure provides a method of treating a
subject in need of botulinum toxin therapy, comprising the step of administering a
therapeutically effective amount of a modified BoNT/E catalytic domain, wherein light
chain residue 224 has been altered, to the subject. The subject in need of botulinum
toxin therapy may suffer from, without limitation, asthma, CF, chronic obstructive
pulmonary, gastric acid efflux and inflammation, immune disorders with a cytokine
component or cancers with a cytokine component.
[0015] While multiple instances are disclosed, still other instances of the present disclosure
will become apparent to those skilled in the art from the following detailed description.
[0016] The detailed descriptions are to be regarded as illustrative in nature and not restrictive.
BRIEF DESCRIPTION OF THE FIGURES
[0017]
Figure 1. K185 of human SNAP23 contributes to the substrate recognition by BoNT/E. (a) Substrate
recognition by LC/E. Two subsites in SNAP25 contribute to substrate binding "B" (Km) and catalysis "AS" (kcat), where the P3, P2, and PI' residues contribute to recognition by LC/E. (b) Sequence
alignment of human SNAP25 (SN25) and human SNAP23 (SN23). (c) (upper panel) modeled
complex structure of LC/ESNAP25 predict the recognition of P site residues of SNAP25
by LC/E. (lower panel) modeled complex structure of LC/E(K224D)-SNAP23 predict the recognition of P site residues of SNAP23 by LC/E(K224D). Models were generated by SWISS-MODEL, using LC/E crystal structure (PDB:3d3x),
and images were generated in PyMol.
Figure 2. Cleavage of SNAP23 by LC/E(K224D). (a) Five µm SNAP23 was incubated with indicated amounts of LC/E(K224D) and subjected to SDS-PAGE (stained gel is shown in insert, SNAP23(152-211) is designated
(SN23(152-211)) and the cleavage product SNAP23(152-186) is designated*. %SNAP23 cleavage
was determined by densitometry. (b) Kinetic constant for LC/E to cleave SNAP25 and
LC/E(K224D) to cleave SNAP23.
Figure 3. Site of SNAP23 cleavage by LC/E(K224D) (a) Five µm SNAP23 was incubated with 2 µm of LC/E(K224D) and subjected to MALDI-TOF Mass Spectrometry. Intensity (100%) on the y-axis was
set to the 2812.5 band and the x-axis represents mass-to-charge units, m/z. (b) SNAP23(I187D) was incubated with the indicated amounts of LC/E(K224D) and subjected to SDS-PAGE. The Coomassie stained gel is shown with the migrations
of LC/E(K224D and SNAP23(I187D) indicated on the left.
Figure 4. Sequence alignment and substrate specificity of LC/E(K224D) and Wt- LC/E on SNAP25 isoforms. (a) Alignment of SNAP23a,b, SNAP25a,b, SNAP29
and SNAP47 (ClustaIW2) in the regions corresponding to SNARE proteins that interact
with the binding region and active sites region of LC/E. Indicated are conserved residues
(*) and similar residues (:, .) among the SNAP25 isoforms. Cleavage site of SNAP25
by LC/E (arrow) and P site resides are indicated. Linear velocity assays of LC/E(K224D) (b) and Wt-LC/E (c) with the indicated isoforms of SNAP25. Five µm SNAP25 isoform
was incubated with the indicated amounts of LC, subjected to SDS-PAGE and gels were
stained with Coomassie. The amount of SNAP25 isoform cleavage was determined by densitometry.
Figure 5. LC/E(K224D) cleaves SNAP23 and inhibits mucin and IL-8 secretion in TGF-α stimulated HeLa cells.
(A) GFP-LC/E(K224D) or GFP-Wt-LC/E were transfected into HeLa cells. After 24h, cell lysates were prepared
and separated by SDS-PAGE and cleavage of SNAP23 was measured by western-blotting
using anti SNAP-23 mouse monoclonal antibody. (B, C). HeLa cells were transfected
with DNA encoding GFP-LC/E(K224D) or GFP-Wt-LC/E. After 24h, cells were washed with serum free MEM medium twice and
secretion was induced by the addition of serum free MEM medium supplemented with 20ng/ml
of TNF-α. After 36h, supernatant were collected and assayed for mucin and IL-8 secretion,
using an ELISA format. The amount of mucin and IL-8 secreted in controls cells was
adjusted to 1.0 and used as a reference for cells treated with TNF-α.
Figure 6. Recombinant LC/E(K224D) cleaves SNAP23 and inhibits mucin and IL-8 secretion in TGF-α stimulated HeLa cells.
HeLa cells were treated with digitonin and then incubated with His-LC/E(K224D) or His-Wt-LC/E (3-Xflag tagged proteins). After an overnight incubation, cells
were washed and then incubated with serum free MEM media supplemented with 20 ng/ml
of TNF-α for 36 h when cell supernatants were collected and cell lysates were prepared.
(a) Cell lysates were subjected to SDS-PAGE and LC/E expression and SNAP23 cleavage
was measured by Western blot, using α-3Xflag antibody and α-SNAP23 antibody, respectively;
* indicates migration of the SNAP23 cleavage product. Culture supernatants were assayed
IL-8 (b) and mucin (c) secretion, using an ELISA, using 1.0 as a reference for cells
treated with recombinant LC/E.
DETAILED DESCRIPTION
I. IN GENERAL
[0018] In the specification and in the claims, the terms "including" and "comprising" are
openended terms and should be interpreted to mean "including, but not limited to....
" These terms encompass the more restrictive terms "consisting essentially of" and
"consisting of."
[0019] As used herein and in the appended claims, the singular forms "a", "an", and "the"
include plural reference unless the context clearly dictates otherwise. As well, the
terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably
herein. It is also to be noted that the terms "comprising", "including", "characterized
by" and "having" can be used interchangeably.
[0020] Unless defined otherwise, all technical and scientific terms used herein have the
same meanings as commonly understood by one of ordinary skill in the art to which
this disclosure belongs.
[0021] All references cited in this specification are to be taken as indicative of the level
of skill in the art. Nothing herein is to be construed as an admission that the invention
is not entitled to antedate such disclosure by virtue of prior invention.
II. THE DISCLOSURE
[0022] The present disclosure provides novel engineered botulinum neurotoxins (BoNTs) that
cleave non-neuronal SNARE proteins and methods of use thereof.
[0023] BoNTs are effective therapeutics for a variety of neurological disorders, such as
strabismus, blepharospasm, hemificial spasm, and cervical dystonia, due to the toxin's
tropism for neurons and specific cleavage of neuronal SNARE proteins. Modifying BoNTs
to bind non-neuronal cells requires retargeting the catalytic activity of BoNTs to
non-neuronal SNARE isoforms to provide effective non-neuronal therapies.
[0024] Here, we extend the substrate specificity of BoNT/E by engineering a catalytic derivative
that cleaves the non-neuronal SNARE protein, SNAP23, as a platform for novel methods
of treating non-neuronal human secretory diseases. By "non-neuronal human secretory
diseases, we mean, for example, diseases or conditions where excessive airway mucus
secretion, mucus hypersecretion, may cause mucus accumulation that is associated with
human clinical conditions such as asthma and chronic obstructive pulmonary disease
where mucus accumulation contributes to respiratory disease. Specifically, we now
report for the first time the engineering of a BoNT/E derivative that cleaves SNAP23,
a non-neuronal SNARE protein.
[0025] SNAP23 mediates vesicle-plasma membrane fusion processes, including secretion of
airway mucus, antibody, insulin, gastric acids, and ions. The mutated BoNT/E light
chain LC/E(K224D) of the presentdisclosure showed extended substrate specificity to
cleave SNAP23, and the natural substrate, SNAP25, but showed no specificity for, and
did not cleave, SNAP29 or SNAP47. Upon direct protein delivery into cultured human
epithelial cells, LC/E(K224D) cleaved endogenous SNAP23, which inhibited secretion
of mucin and IL-8. These studies show for the first time the feasibility of genetically
modifying BoNTs to target a non-neuronal SNARE protein for novel methods of treating
human hyper-secretion diseases, such as asthma and chronic obstructive pulmonary disease.
[0026] In one instance, the present disclosure is a preparation of an engineered catalytic
domain of botulinum neurotoxin E that is capable of specifically cleaving SNAP23.
Preferably, the toxin cleaves SNAP25 but not SNAP29 or SNAP 47. The LC/E(K224D) can
cleave both SNAP25 and SNAP23, but at ∼10 fold lower than Wt-LC/E can cleave SNAP25.
[0027] In a preferred instance, the modified botulinum neurotoxin E of the present disclosure
has a mutation at light chain residue 224 or a residue corresponding to residue 224.
By "residue 224" we mean the lysine at position numbered 224 of SEQ ID NO: 7. SEQ
ID NO: 7 is the protein conversion of the DNA sequence of SEQ ID NO: 1 absent the
first methionine residue. By "residue corresponding to residue 224" we mean the lysine
within the motif of residues 210-240 of SEQ ID NO: 7. Specifically, we mean the highlighted
lysine within residues MHELIHSLHGLYGAKGITTKYTITQKQNPLI (SEQ ID NO: 8). Other botulinum
type E subtypes have this same corresponding residue and motif, although the numbering
may not be identical among subtypes. However, the motif residues will be at least
90% corresponding to the motif of SEQ ID NO: 8.
[0028] One may find an exemplary sequence of botulinum toxin E subtype Beluga light chain
at GeneBank with accession number X62089 (SEQ ID NO: 1). We anticipate that other
subtypes of serotype E could be used as template for engineering LC that can cleave
SNAP23.One would modify residue numbered 224 of any botulinum toxin E subtype in the
same manner as disclosed within the present disclosure.
[0029] In a preferred instance, the present disclosure is a preparation of a modified botulinum
toxin E with a mutation at a light chain residue 224 which cleaves SNAP23. Another
instance of the present disclosure is a preparation of botulinum neurotoxin E light
chain with a mutation at light chain residue 224 which cleaves SNAP23. Another instance
of the present disclosure is a preparation of botulinum neurotoxin catalytic domain
(residues 1 through 400) with a mutation at light chain residue 224 which cleaves
SNAP23. Another instance of the present disclosure is a truncated fragment of the
catalytic domain, comprising at least residues 1 to 390, which comprises a modified
residue 224 which cleaves SNAP23. By "modified catalytic domain" we mean to include
all forms, including humanized forms, of the catalytic domain of L/C BoNT with a modification
at residue 224.
[0030] Another preferred instance is a mutated botulinum neurotoxin E light chain or modified
catalytic domain fused to another peptide, as described below, for appropriate therapeutic
methods.
[0031] In another instance, the present disclosure is a DNA sequence encoding the engineered
botulinum neurotoxin or the modified catalytic domain described herein.
[0032] An exemplary reference to the sequences of SNAP23, SNAP25, SNAP29 and SNAP47 can
be found at GeneBank with accession numbers CR457212 (SEQ ID NO: 2), NM_130811 (SEQ
ID NO: 3), CR456582 (SEQ ID NO: 4) and BC011145 (SEQ ID. NO: 5).
[0033] In other instances of the disclosure, other mutations of residue 224 would also be
suitable. The mutation within LC/E that is being protected is K224D, which recognizes
the P2 residue of SNAP23, which is a lysine. We anticipate that a glutamic acid mutation
at K224 would also yield a functional LC/E that can cleave SNAP23. We generated a
K224A mutation that has the ability to cleave both SNAP23 and SNAP25, but with less
efficiency than K224D, which indicates that charge and size of the R-group influence
cleavage efficiency. Thus, we anticipate that other residue replacements, such as
replacing the lysine at residue 224 of SEQ ID NO: 7 with glutamic acid, may also provide
a mutated LC/E with the ability to cleave SNAP23.
[0034] In other instances, we further anticipate that additional residue replacements, alone
or in combination with the mutation of K224D of the present invention, may yield a
LC/E that has the ability to cleave SNAP 23 and not SNAP 25.
[0035] Methods of Treatment. In other instances, the present disclosure provides novel methods of treating a subject
requiring treatment with a botulinum toxin. In one instance, the present disclosure
provide methods of treating a subject suffering from a human secretory disease by
administering a therapeutically effective amount of the modified catalytic domain
as described above.
[0036] By "subject" we mean any person requiring treatment with botulinum toxin. By "treating"
or "treatment", we mean the management and care of a subject for the purpose of combating
the disease, condition, or disorder. The terms embrace both preventative, i.e., prophylactic,
and palliative treatment. Treating includes the administration of a compound of present
disclosure to prevent the onset of the symptoms or complications, alleviating the
symptoms or complications, or eliminating the disease, condition, or disorder. In
one instance, we envision the method of the presentdisclosure reducing symptoms by
at least 20 to 50 percent. We envision treatment occurring on a regular basis until
symptoms are reversed. For instance, in one instance, treatment would occur daily,
weekly or monthly, as needed.
[0037] By "therapeutically effective amount" we mean amount of a compound that, when administered
to a subject for treating a disease, is sufficient to effect such treatment for the
disease. The "therapeutically effective amount" will vary depending on the compound,
the disease state being treated, the severity or the disease treated, the age and
relative health of the subject, the route and form of administration, the judgment
of the attending medical or veterinary practitioner, and other factors as known to
one of skill in the art (40). This method would involve administering to the subject
the modified catalytic domain of the engineered botulinum toxin E of the present disclosure.
By "administering" we mean delivering the engineered BoNT of the present disclosure
to the subject. Although the preferable form of the catalytic domain is the botulinum
toxin E light chain, the catalytic domain may include forms that are shorter than
the light chain, such as the C-terminal truncation mutants of the catalytic domain
to residues ∼1-390 residues (41), and forms that are larger than the light chain and
include the translocation domain or fragments of the translocation domain up to ∼residue
F
1200 which includes the N terminus of the HCR domain. In one instance, we have engineered
a 1-400 which has good solubility and activity.
[0038] In one instance, the catalytic domain is complexed, either through covalent bonds
or attached in some other way, such as cross-linking, to a targeting molecule (targeting
system). The targeting molecule is adapted to target the toxin to a cell-surface receptor
of interest.
[0039] As described above, we envision several forms of the catalytic domain as being effective
potential delivery platforms for the K224D mutation. For instance, protein solubility
may vary with the application and the chimera that is engineered, therefore different
sized catalytic domains may be more useful in specific applications. We anticipate
that the amount of effective delivery platform will be similar to the amounts of BoNT
used in clinical therapy and that the amount of effective delivery can be fractional
based upon the catalytic nature of LC/E(K224D).
[0040] In other instances, a suitable delivery system will, in one instance, target specific
cell types or cell surface receptors that are internalized and deliver LC/E into the
cytoplasm, such as an antibody or a growth factor. Potential cell surface receptors
that can be targeted include tissue specific growth factor receptors, which could
vary with the disease that is targeted. For example, one of skill would understand
based on materials known to the field how to target CD22 in hairy cell leukemia (42).
[0041] Alternatively, a lipid-based directed protein delivery system could be used to deliver
LC/E directly into the host cytosol. For instance, a targeted-liposome delivery which
delivers either the catalytic domain protein directly or DNA encoding the LC/E(K224D)
protein (39) are also effective methods of delivering the engineered BoNT/E of the
present disclosure to treat various diseases. For instance, lipid based delivery system
that assemble nano-particles for efficient delivery and reduced immunogenicity should
prove useful as targeting vehicles (43). While the different delivery methods may
not have different advantages based on the disease being targeted, the specificity
of the delivery system will be a critical factor in selecting a specific delivery
system.
[0042] To optimize the potency, we envision humanizing the modified catalytic domain using
one of several possible approaches known to the art (34, 35).
[0043] Several delivery systems are envisioned for the therapeutic delivery of LC/E(K224D),
or another suitable toxin, to a subject requiring treatment for, for instance, a non-secretory
or hyper-secretory disease. Example delivery systems include, without limitation,
single chain protein chimeras where the modified catalytic domain, preferably LC/E(K224D),
is fused either at the DNA level or by protein-receptor cross-linking (36) or bipartate
protein delivery systems where the catalytic domain, preferably LC/E(K224D), is linked
to a fusion composed of a di-protein delivery system (37, 38). For example, the gene
encoding LC/E(K224D) could be genetically fused to the gene encoding the epidermal
growth factor to target non-small-cell lung cancer.
[0044] The table below describes some of the appropriate uses for the engineered toxin of
the presentdisclosure and appropriate targets for the targeting domain.
TABLE 1: Receptor/Disease
LC/E(K224D)-Receptor |
Disease |
Lung epithelium cell specific receptor |
Asthma, CF, chronic obstructive pulmonary disease |
Gastric specific receptor |
Gastric acid efflux and inflammation |
Mast cell specific receptor |
Allergic rhinitis, Hemophagocytic lymphohistiocytosis, and Chronic urticaria |
Cancer specific receptor |
Renal cell carcinoma, Nonsmall cell lung cancer, and gastric cancer, Epithelial ovarian
cancer, and Estrogen receptor (ER)-positive breast cancer |
[0045] Kits. In an alternate instance of the disclosure, a kit for treating a subject with the
modified catalytic domain of the present disclosure is provided. In one instance,
the kit comprises a form of the engineered BoNT of the present disclosure and instructions
for use. In one instance, the modified catalytic domain of the present disclosure
is formulated, delivered and stored for use in physiologic conditions. In a preferred
instance, the kit also comprises a targeting system. The modified catalytic domain
is either already attached to the targeting system or the kit contains the targeting
system with instructions for attachment. In alternate instances, the kit comprises
DNA encoding LC/E(K224D) that can be used to engineer fusion proteins to specific
tissue specific targeting molecules.
[0046] By "instructions for use" we mean a publication, a recording, a diagram, or any other
medium of expression which is used to communicate the usefulness of the disclosure
for one of the purposes set forth herein. The instructional material of the kit can,
for example, be affixed to a container which contains the present disclosure or be
shipped together with a container which contains the disclosure. Alternatively, the
instructional material can be shipped separately from the container or provided on
an electronically accessible form on an internet website with the intention that the
instructional material and the biocompatible hydrogel be used cooperatively by the
recipient.
III. EXAMPLES
[0047] The following examples are offered for illustrative purposes only and are not intended
to limit the scope of the present invention in any way. Indeed, various modifications
of the invention in addition to those shown and described herein will become apparent
to those skilled in the art from the foregoing description and the following examples
and fall within the scope of the appended claims.
A. METHODS AND MATERIALS.
[0048] Molecular modeling. Molecular modeling was performed using SWISS-MODEL. The structure of the LC/E-SNAP25
(146-202) complex was obtained as described (19), using the crystal structure of LC/E
(PDB:3d3x). The structure of LC/E(K
224D)-SNAP23 was modeled using LC/E-SNAP25 complex structure as a template, using PyMol™.
Data presented are the average of experiments performed at least 3 times.
[0049] Plasmid construction and protein expression. BoNT LC/E expression vector was constructed by amplifying DNA encoding LC/E (1-400)
of Clostridium botulinum serotype E Beluga (SEQ ID NO: 1) and subcloning into pET-15b.
For transfection experiments, LC/E(1-400) was also subcloned into pEGFP vector to
generate an EGFP-LC/E (1-400) fusion protein expressed under the CMV promoter. Expression
vectors for SNAP23(152-211), SNAP29(202-259) and SNAP47(406-464), the protein equivalents
of SNAP25(145-206), were constructed by PCR amplifying and cDNA template: human SNAP23
(ATCC 2900640, SEQ ID NO:2), SNAP29(ATCC 10700609, SEQ ID NO: 4) and SNAP47(ATCC 10468826,
SEQ ID NO: 5) and subcloning into pGEX-2T. Site-directed mutagenesis was performed
using QuickChange (Stratagene). Protein expression and purification were performed
as previously described (32).
[0050] Cleavage of SNARE protein by LC/E and LC/E(K224D). Cleavage of SNARE protein was performed as described.
[0051] Linear velocity reaction: Reactions contained (10 µl): 5 µM human SNARE proteins, 10mM Tris-HCl (ph 7.6) with
20 mM NaCl, and the indicated amounts of LC/E and LC/E(K
224D). Reactions were incubated for 10 min at 37°C, subjected to SDS-PAGE and gels were
stained with Coomassie. The amount of SNARE protein cleavage was determined by densitometry.
[0052] Kinetic parameters: K
m and k
cat determinations were made for Wt-LC/E and LC/E derivatives using SNAP25 isoforms.
LC concentrations were adjusted to cleave <10% substrate at several concentrations
of substrate (1.5∼18 µM SNARE protein). Reactions were carried out at 37°C for 10
min, subjected to SDS-PAGE and the amount of cleaved product was calculated by densitometry.
Reaction velocity versus substrate concentration was fit to the Michaelis-Menten equation,
using Lineweaver-Burk plots, using SigmaPlot IX (Chicago, IL).
[0053] LC/E and LC/E(K224D) activity in human cultured epithelial cells. HeLa cells were cultured in 6 well plates in MEM supplemented with 10% newborn calf
serum. Sub-confluent cells were transfected with 0.5 or 1.0 µg of indicated plasmid
using Lipofectamine LTX (GIBCO/BRL).
[0054] Protein delivery. Protein delivery was performed as described (33) with modification. HeLa cells were
permeabilized with 1 ml/well of permeabilization buffer containing 30 µM digitonin
for 7 min and the incubated in permeabilization buffer with and without the indicated
LC.
[0055] Protein Secretion Assays: After an overnight incubation, transfected and protein delivered cells were incubated
in 2m1 serum free MEM containing 20 ng/ml of TNF-α. After 36h, 1.5 ml of supernatant
was collected, centrifuged at 13,000 g for 1 min, and assayed for secreted mucin and
IL-8, using ELISA. Supernatants (150 µl) were mixed with 50 µl of 0.2 M Na
2CO
3 (pH 9.6) and added to 96-well plates and incubated overnight at 4°C. Plates were
washed and locked with 50 mM Na
2CO
3 (pH 9.6) buffer containing 1% (w/v) BSA. Plates were washed and incubated with 100
µl α-mucin IgG (1/200 dilution, Abcam) or α-IL-8 IgG (1/200 dilution, Abcam) for 1h
at RT. Plates were washed 3 and incubated with α-mouse Horse Radish Peroxidase-conjugate
antibody (1:10,000 dilution, Pierce) for 1h at RT. Plates were washed and developed
with 100 µl of Ultra-TMB (Pierce) for 20 min at RT and quenched with 100 µl of 1M
H
2SO
4. A
450 was expressed as fraction relative to secreted mucin or IL-8 in control supernatants.
[0056] Cleavage of SNAP23: lysates from cells incubated with TNF-α for 36h were assayed for LC/E and LC/E(K
224D)-mediated a cleavage of endogenous SNAP23, using α-SNAP23 mouse IgG (Abcam, Cambridge,
MA) by Western blot analysis (19).
[0057] Delivery Systems. The mutated BoNT was administered to a subject using the following delivery methods:
Target-specific Cell Receptor: One skilled in the art of molecular biology would be able to engineer chimeras of
LC/E(K224D) fused to targeting molecules, using polymerase chain reaction-like protocols.
Lipid-based: One skilled in the use of lipid based delivery systems would be able to develop lipid
LC/E(K224D) ratios for the efficient internalization of the LC by utilizing a lipid
to protein matrix.
[0058] Methods of Treatment. The engineered BoNT of the present disclosure was administered to a subject to treat,
in one instance, hyper-secretory and non-secretory diseases. One of skill would identify
a subject for treatment with the engineered BoNT, administer the treatment, monitor
the results, and determine the effectiveness of the treatment, following strategies
utilized for the development of immunotoxin therapy in the treatment of hairy cell
leukemia.
B. RESULTS.
[0059] Previous studies identified residues 167-186 as the minimal, optimal peptide of SNAP25,
a 206 amino acid protein for LC/E in vitro cleavage (19). SNAP25 (167-186) comprises
two sub-sites that include a substrate binding "B" region and an active site "AS"
region (Fig. 1a). LC/E recognizes the P3 residue to facilitate alignment of the P2
and P1' residues of SNAP25. The S1' pocket of LC/E is formed by F
191, T
159, and T
208 with hydrophobic interactions between F
191 of LC/E and the P1' residue I
181 of SNAP25 (20). The basic S2 pocket contains K
224, which recognizes the P2 residue D
179, through a predicted salt bridge. Docking the P2 and P1' residues of SNAP25 into
the active site pockets of LC/E aligns the scissile bond for cleavage (19, 20).
[0060] BoNT/E was known as not cleaving human SNAP23 (8), providing a framework of defining
SNAP isoforms specificity of the BoNTs. Many of the residues that contributed to LC/E
recognition of SNAP25 were conserved in human SNAP
23, except T
173/A
179, D
179/K
185, M
182/T
188 and E
183/D
189, respectively (Fig. 1b). T
173 in SNAP25 played only a limited contribution for LC/E substrate recognition (20)
and only main chain interactions of M
182-D
186 contributed to LC/E substrate recognition. Thus, the T
173/A
179, D
179/K
185, M
182/T
188 and E
183/D
189 differences between SNAP25 and SNAP23 did not appear to contribute to inability of
cle to cleave SNAP23. In contrast, the P2 residue of SNAP25, D
179, is recognized by the basic S2 pocket of cle via the basic residue, K
224, which contributes to cle substrate recognition (Fig. 1c, upper panel). Accordingly,
the inventors examined whether the salt bridge between K
224 of LC/E and D
179 of SNAP25 contributes the ability of LC/E to cleave SNAP25 and that charge repulsion
between K
224 of LC/E and the P2 residue of SNAP23, K
185, contributes to the inability ofLC/E to cleave SNAP23. To test this hypothesis, a
point mutation, K
224D, was introduced into LC/E and tested for the ability to cleave human SNAP23 (Fig.
1c, lower panel).
[0061] LC/E(K
224D) cleaved human SNAP23 with a K
m of ∼ 3 µM and k
cat of ∼17 S
-1 (Fig. 2), with 2-fold of the K
m and 5-fold of the k
cat of LC/E for the cleavage of human SNAP25. The specific activity for the cleavage
of SNAP23 by LC/E(K
224D) was similar to the cleavage of VAMP-2 by the B serotype of BoNT and ∼10 fold faster
for the cleavage of VAMP-2 by tetanus toxin (21, 22). The site that LC/E(K
224D) cleaved SNAP23 was identified by MALDI-TOF MS were a major peak with an m/z value
of 2812.5 was identified in a reaction mixture that contained SNAP23 and LC/E(K
224D) (Fig. 3a), corresponding to the C-terminal 25 amino acid of human SNAP23, IKRITDKADTNRDRIDIANARAKKLIDS
(SEQ ID NO: 6). This indicated that LC/E(K
224D) cleaved SNAP23 between
186R-I
187. The determination that LC/E(K
224D) did not cleave SNAP23(I
187D) (Fig. 3b) supported that LC/E(K
224D) cleaved human SNAP23 between residues
186R-I
187.
[0062] SNAP25 isoforms include SNAP25a, SNAP25b, SNAP23a, SNAP23b, SNAP29 and SNAP47 (23,
24). SNAP23 and SNAP25 mediate synaptic membrane fashion in non-neuronal and neuronal
cells, respectively, while SNAP29 and SNAP47 have not been implicated in membrane
fusion events. SNAP29 was shown to inhibit SNARE disassembly and was implicated in
synaptic transmission (25). While the function of SNAP47 is not clear, SNAP47 can
substitute for SNAP25 in SNARE complex formation and proteoliposome fashion. The substrate
specificity of LC/E(K
224D) on SNAP25 isoforms including SNAP23a, SNAP25b, SNAP29 and SNAP47 were tested. SNAP23b
and SNAP25a were not tested because the a-b isoforms of SNAP23 and SNAP25 were identical
and the LC/E substrate recognition region (Fig. 4a). LC/E(K
224D) showed similar activity on both SNAP23 and SNAP25 (Fig. 4b), but did not cleave
SNAP29 and SNAP47. Wt-LC/E cleaved SNAP25 (Fig. 4c), but not the other SNAP25 isoforms.
The specificity of another LC/E K
224 mutation (K
224A) on SNAP23 and SNAP25 was also characterized. LC/E(K
224A) cleaved SNAP23 and SNAP25 with similar efficiencies, but at a slower rate than
LC/E(K
224D) (data not shown).
[0063] Next, the ability of LC/E(K
224D) to cleave endogenous SNAP23 in HeLa cells was tested. While a role for SNAP23 in
constitutive exocytosis is not apparent (26), SNAP23 contributes to regulated exocytosis
(27). Transfection of ∼60% of HeLa cell population with LC/E(K
224D) resulted in the cleavage ∼45% of the SNAP23, while SNAP23 cleavage was not detected
when HeLa cells were transfected with Wt-LC/E or a no plasmid control (Fig. 5). This
indicated that LC/E(K
224D), but not Wt-LC/E, cleaved endogenous SNAP23 in cultured cells. The effect of SNAP23
cleavage on HeLa cell secretion was tested in LC/E(K
224D)-transfected HeLa cells by analyzing TNF-α- mediated mucin and IL-8 secretion. Control
HeLa cells secreted mucin and IL-8 upon addition of TNF-α, while LC/E(K
224D)-transfected HeLa cells showed reduced mucin and IL-8 secretion (Fig. 5b and c).
The inhibition was specific, since Wt-LC/E-transfected HeLa cells showed the same
amount of mucin and IL-8 secretion as control cells and did not cleave endogenous
SNAP23 (Fig. 5).
[0064] To test the feasibility of utilizing LC/E(K
224D) as a protein therapy, recombinant LC/E(K
224D) was delivered into HeLa cells, using digitonin. Recombinant LC/E(K
224D) cleaved endogenous SNAP23 (Fig. 6), which inhibited TNF-α-mediated mucin and IL-8
secretion (Fig. 6b and c). Digitonin treatment also delivered Wt-LC/E into HeLa cells,
but Wt-LC/E-treated HeLa cells, did not show detectable inhibition of mucin and IL-8
secretion and did not cleave endogenous SNAP23 (Fig. 6). This supports a role for
SNAP23 in regulated exocytosis pathways in epithelial cells and indicates the utility
of LC/E(K
224D) as a research tool to study SNAP23-regulated exocytosis (27).
C. DISCUSSION.
[0065] Understanding of substrate specificity of botulinum neurotoxins has enabled the engineering
of a novel light chain derivative of BoNT/E with extended substrate specificity, providing
a proof of principle to extend the clinical potential of BoNT therapy beyond neurological
applications. While airway mucus protects the epithelial lining by entrapping and
clearing foreign debris, bacteria, and viruses from the airway by ciliary movement,
a process termed mucociliary clearance (17, 18), excessive airway mucus secretion,
mucus hypersecretion, may cause mucus accumulation that is associated with human clinical
conditions such as asthma and chronic obstructive pulmonary disease where mucus accumulation
contributes to respiratory diseases. Mucus secretion is a regulated process coordinated
by several molecules, including SNARE proteins, myristoylated alanine-rich C kinase
substrate (MARCKS), and Munc proteins, which coordinate the docking of mucin containing
vesicles with the secretory cell plasma membrane for exocytosis (17, 18). Targeting
SNAP23 by a substrate modified BoNT may reduce the secretion processes of hypersecretion
syndromes. A SNAP23-specific BoNT may also be targeted for other therapeutic applications
that include diabetes and inflammatory and immune disorders which also include a hypersecretory
component (28, 29).
[0066] Alignment and biochemical analyses allow prediction of the mechanism for the catalytic
activity of Wt-LC/E and LC/E(K224D) on SNAP25 isoforms. The low overall homology within
the active site regions of SNAP29 and SNAP47 to SNAP25 and the lack of an isoleucine
at the P1' site explain the inability of Wt-LC/E and LC/E(K224D) to cleave SNAP29
and SNAP47. In contrast, the overall homology between SNAP23 and SNAP25 is high, except
at the P2, P2' and P3' residues with the most dramatic change at the P2 residue where
SNAP25 contains an aspartate and SNAP23 contains a lysine. Thus, one reason for the
inability of Wt- LC/E to cleave SNAP23 may be due to the electrostatic repulsion of
the P2 residue lysine within SNAP23 by K
224 of LC/E. This may destabilize the S2 pocket and affect alignment of the P1' residue
into the S1' pocket. The ability of LC/E(K224D) to cleave SNAP23 may be due to the
introduction of a salt bridge between the P2 residue Lys of SNAP23 and the mutated
S2 pocket residue D
224. LC/E(K224D) also retained the ability to cleave SNAP25, although at a rate that
was ∼10 fold slower than Wt-LC/E. This suggests that the repulsion between the P2
residue aspartate of SNAP25 and the mutated S2 pocket residue D
224 was not sufficient to inhibit sessile bond cleavage by LC/E(K
224D).
[0067] Since LC/E(K224A) cleaved SNAP25 and SNAP23, but at a reduced rate relative to LC/K224D,
both charge and size of the R-group at residue 224 contribute to optimal scissile
bond cleavage. Overall, the biochemical properties of LC/E and LC/E-K
224 derivatives are consistent with P2 residue-S2 pocket residue interactions contributing
to the efficiency of sessile bond cleavage, while not binding SNAP29 and SNAP47. In
contrast, the overall homology between SNAP23 and SNAP25 is high, except at the P2,
P2' and P3' residues with the most dramatic change at the P2 residue where SNAP25
contains an aspartate and SNAP23 contains a lysine. Thus, one reason for the inability
of Wt-LC/E to cleave SNAP23 may be due to electrostatic repulsion of the P2 residue
lysine within SNAP23 by K
224 of LC/E. This may destabilize the S2 pocket and affect alignment of the PI' residue
into the S1' pocket.
[0068] The ability of LC/E(K224D) to cleave SNAP23 may be due to the introduction a salt
bridge between the P2 residue Lys of SNAP23 and the mutated S2 pocket residue D
224. LC/E(K224D) also retained the ability to cleave SNAP25, although at a rate that
was ∼10 fold slower than Wt-LC/E. This suggests that the repulsion between the P2
residue aspartate of SNAP25 and the mutated S2 pocket residue D
224 was not sufficient to inhibit sessile bond cleavage by LC/E(K
224D). Since LC/E(K
224A) cleaved SNAP25 and SNAP23, but at a reduced rate relative to LC/EK
224D, both charge and size of the R-group at residue 224 contribute to optimal scissile
bond cleavage. Overall, the biochemical properties of LC/E and LC/E-K
224 derivatives are consistent with P2 residue-S2 pocket residue interactions contributing
to the efficiency of sessile bond cleavage. While the ability of native LC/E to bind
SNAP23 has not been determined, kinetic values for LC/E and SNAP25 and LC/E(K
224D) and SNAP23 are within 2-fold, indicating similar binding affinities.
[0069] Alignment of SNAP25 and SNAP23 within the LC/E binding region (Fig.1) is nearly identical
with 7 of 8 residues identical and the non-identical pair being T:A; a conserved substitution
pair, which also supports similar binding affinities of LC/E for SNAP25 and SNAP23.
SNARE proteins are key proteins in membrane fusion and trafficking within neuronal
secretary pathways (9). The use of BoNT has contributed to the understanding vesicle
fusion and neurotransmitter release mechanisms in neuronal cells. The ability of a
BoNT derivative to cleave non-neurological SNAREs may provide a useful tool to investigate
intracellular vesicular trafficking and the mechanism of membrane fusion in nonneuronal
systems.
[0070] Although BoNT/A could be considered the logical serotype to be engineered for novel
applications due to its wide clinical applications, analysis of the mechanisms of
SNAP25 recognition indicate that LC/A requires a longer substrate for optimal SNAP25
recognition with a greater number of residue interactions than LC/E (20). The less-complex
SNAP25-LC/E interactions make BoNT/E amenable for engineering to modify substrate
recognition. In addition, alignment of human SNAP25 and SNAP23 showed that these proteins
had a high level of homology at the P3 and PI' sites that are involved in SNAP25 recognition
by LC/E. Thus, BoNT/E is a useful platform to engineer mutations that effect SNARE
protein recognition. The successful delivery of LC/E(K
224D) into cells to inhibit IL-8 and mucin secretion supports a role for LC/E(K
224D) as a research tool and also shows the potential for therapy to regulate human hypersecretion
diseases such as asthma and inflammatory diseases. The therapeutic specificity of
LC/E(K
224D) would be based upon the receptor binding component, as described for toxin chimeras,
such as diphtheria toxin A fragment-IL2 (30) and Exotoxin A fragment-IgG variable
region fragment (31). In conclusion, the current study shows proof of principle for
altered substrate specificity to extend the application of BoNTs beyond neurological
inflictions.
[0071] It should be noted that the above description, attached figures and their descriptions
are intended to be illustrative and not limiting of this invention. and variations
are within the contemplation hereof.
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SEQUENCE LISTING
[0073]
<110> Medical college of Wisconsin
<120> ENGINEERED BOTULINUM NEUROTOXIN
<130> 650053.00177
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